2094
EPHRAIM KATCHALSKI, ISAAC GROSSFIELD AND MAXFRANKEL
Vol. 70
[CONTRIBUTION FROM THE LABORATORY OF HIGHMOLECULAR CHBhtISTRY, THE HEBREW UNIVERSITY]
Poly-condensation of a-Amino Acid Derivatives. III. Poly-lysine BY EPHRAIM QTCHALSKI,
ISAAC GROSSFELD AND MAXFRANKEL
In the previous papers of this series we have necessary, as it was found that while the freshly described the preparation and properties of some prepared substance undergoes rapid polymerizapolymers of glycine2”and d,Z alanine.2b As an ex- tion a t its melting point (loo’), the same subtension of this work the study of the polymeriza- stance after having been allowed to stand for tion of poly-functional a-amino acids was under- several weeks did not polymerize even a t higher taken. In the present paper the formation of temperatures (160-170 ), but decomposed slowly poly-lysine by the polymerization of a 1-lysine under such conditions. In this connection the obderivative is described. servation of Bergmann, Zervas and ROSS,‘may The following considerations had to be taken be recalled that the melting point of freshly preinto account in the choice of a suitable monomer pared e-carbobenzoxy-a-carboxyl-1-lysine anhyfor polymerization: (a) the e-amino group of the dride rises on standing several months to above lysine had to be masked in order to prevent its 250’. Our attempts to recover the e-carbobenparticipation in the polymerization and thus to zoxy-a-carboxyl-1-lysineanhydride with a meltinsure that all the -CONHbonds in the poly- ing point of 100’ from the preparation having a mer are derived from the a-amino groups of the high melting point by recrystallization were unmonomer. The e-amino group was masked by successfulas no solvent for the latter was found. During search for other precautions to be taken Bergmann’s method of introducing the carbobenzoxy group.; (b) the monomer must consist of a in the polymerization of e-carbobenzoxy-a-carlysine derivative having a pronounced tendency boxyl-1-lysine anhydride, the following findings to polymerize. e-Carbobenzoxylysine itself is were useful: (a) pure a,€-di-carbobenzoxy-1-lycomparatively stable owing to its zwitterionic sine, as well as pure carbobenzoxy-glycine, give no structure, while its methyl ester yields mainly amino N values on using Van Slyke’s6manometthe corresponding diketopiperazine on heating;a ric method for amino N determination. (b) e-Carbobenzoxy-1-lysine yields practically both these lysine derivatives did not appear therefore to be the proper starting material. A the theoretical amount (96%) of carboxyl nitrogen more suitable monomer was eventually found in in Van Slyke, MacFadyen and Hamilton ninhye-carbobenzoxy-a-carboxyl-lysine anhydride.‘ It drin-COz method’ when the reaction is carried out was found that this substance polymerizes on a t pH 2.5. (c) e-Carbobenzoxy-a-carboxyl-1-lysine anhyheating with the evolution of carbon dioxide in a manner similar to the polymerization of N-car- dride evolved an amount of carbon dioxide equivalent only to about 50% of its total a-nitrogen when boxyl anhydrides of other amino acids. The steps of preparation of poly-lysine from analyzed by the usual ninhydrin-COz method. the chosen monomer are summarized in the first The samples were boiled, after addition of citpart of the scheme: c-carbobenzoxy-a-carboxyl- rate buffer, pH 2.5, for thirty seconds to remove 1-lysine anhydride (I), yields on heating, under performed carbon dioxide. The solution was the catalytic action of water, poly-carbobenzoxy- chilled and the N-carboxyl determination camed lysine (11), carbon dioxide being evolved. The out as usual after addition of ninhydrin. carbobenzoxy groups of (11) were removed by Finding (a) shows that during the carrying out phosphonium iodide, and poly-lysine hydriodide of the amino nitrogen determinations, no cleavage of the NH-carbobenzoxy bond, leading to the (111) obtained. For each polymerization experiment, the start- liberation of free NHa groups, occurs. It is thus ing material, e-carbobenzoxy-a-carboxyl-1-lysine possible to determine the free amino-N in mateanhydride, had to be freshly prepared from a,€- rials containing NH-carbobenzoxy bonds, without carbobenzoxy-1-lysine kept in stock. This was fear that additional free amino groups, not present in the original material will be liberated on analy(1) A manuscript under this title was received from these authors on January 21,1946, for publication in the Journal. It wan returned sis. Finding (b) permits the determination of eto them on February 21, 1946, for revision and further data.-The carbobenzoxy-lysine in the presence of poly-carEditor. bobenzoxy-lysine. Furthermore, in view of (b) (2) (a) Frankel and Katchalski, THISJOURNAL, 64, 2264 (1942); and (c), it is clear that high values of carboxyl-N (b) Frankel and Katchalski, ibid., 64, 2268 (1942). (3) Katchalski, Grossfeld and Frankel, ibid., 68, 879 (1946). (determined according to the ninhydrin-COz (4) Bergmann, Zervas and Ross, J . Biol. Chcm., 111,245 (1935). method) indicate the presence of a considerable (5 Leuchs, Bcr., 89, 857 (1906); Leuchs and Manasse, ibid., 40, amount of either or both e-carbobenzoxy lysine 324 (1907); Leuchs and Geiger, ibid., 41, 1721 (1908); Curtius and Sieber, ibid., 55, 1543 (1922); Wessely, 2.phyriol. Chcm., 146, and e-carbobenzoxy-a-carboxyl lysine anhydride. 72 (1925): Sigmund and Wessely, ibid., 157, 91 (1928); Wessely This conclusion enables us to estimate roughly the and Sigmund, ibid., 159, 102 (1926); Wessely and John, ibid., amount of “monomer” derivatives in the prepara170, 38 (1927); Meyer and Go, Help. Chim. Acta. l?, 1488 (1934);
Q
G o nnd Tan& BnU. Chsm. SOC.Japan, 14, 610 (1939); Woodward aad Schramm, TAISJOURNAL, 69, 1561 (1947).
(6) Van Slyke, J . Bid. Chcm., 88, 425 (1029). (7) Van Slyke, MacFadyen and Hamilton, ibid., 141, 671 (1941).
June, 1948
POLY-CONDENSATION OF
~ - A M ~ ACID I N O DERIVATIVES : POLY-LYSINE
2095
oc--0
Cbzo
OCOCHaCJls
-
-OH
O,N~HN--CH-CO-
I (CH2)4 I
HI.HzN-CH-CO-
CaHa(NW2F
I I
CHd4 "*HI
-1
O ~ N ~ H N - ~ H - C O O H (CH2)r
I
HIN-CH-COOH
+
"
(n - 1)
I I
(CHd4
NH
(VI
(VI)
leum ether and then dried for several days over sulfuric acid. The dried anhydride was left to polymerize in an open vessel at 105' for twenty-four hours. (b) Obtained from e-carbobenzoxya-carboxyl-l-lysine anhydride recrystallized twice from ethyl acetate and petroleum ether and dried in a 20-mm. vacuum over phosphorus pentoxide for forty-five minutes. Polymerization was carried out in the dryiig apparatus by raising the temperature to 104 O , and maintaining this temperature for several hours. TABLEI (c) Obtained from e-carbobenzoxy-a-carboxyl-l-lyANALYTICAL DATAON PREPARATIONS OBTAINED ON HEAT-sine anhydride recrystallized three times from ethyl acetate and petroleum ether, and introduced immediately ING ~!ARROBENZOXY-CY-CARBOXYI,-LYSINE ANHYDRIDE UNafter the last recrystallization into a high vacuum apDER VARIOUS CONDITIONS paratus containing phosphorus pentoxide and liquid air --big. of element (or group) per 100 mg. preparationtrap. The monomer was dried in this apparatus for thret Amino Carboxyl Total hours at 50" and then the temperature raised to 104 Prepn. N N N C H Remarks for two hours. A high vacuum (10-4 mm.) was mainTransparent brit- tained throughout. The polymeric preparation repretle film, readily sents a hard, colorless transparent substance. (d) The preparation of this product is described in soluble in cold (a) 2.00 1.09 10.7 63.5 6.9 detad under the heading preparation of poly-carbobenzoxy(b) 1.08 0.092 10.7 glacial acetic lysine (n average = 32 carbobenzoxy-lysine units) acid, insoluble in water tions of polymerization. Table I1 contains data (Transparent film concerning products derived on reduction of the soluble in hot corresponding preparations described in Table I. (c) 0.22 0.02ij 10.8 04.2 6 . 8 Table I shows clearly that the purification of (d) 0.17 0.01'7 1.3 63.9 7 . 1 the e-carbobenzoxy-a-carboxyl-lysine anhydride by recrystallization from ethyl acetate and pe[ in water (a) Obtained from e-carbobenzoxy-a-carboxyl-1-lysine troleum ether and the thorough drying of freshly anhydride recrystallized once from ethyl acetate and petro- prepared monomer leads on heating to prepara-
tions obtained on heating r-carbobenzoxy-a-cboxyl lysine anhydride, even before removal of the carbenzoxy groups. In Table I we summarize various analfical data concerning preparations obtained on heating e-carbobenzoxy-a-carboxyl-lysine anhydride, purified in different ways and using different condi-
. . ...
I
EPHRAIM KATCHALSKI, ISAACGROSSFELD AND MAXFRANKEL
2096
Vol. 70
TABLE I1 ANALYTICAL I>ATA ON PRODUCTS DERIVED FROM THE CORRESPONDING PREPARATIONS DESCRIBED IN TABLE I, ON REDUCTION WITH PHOSPHONIUM IODIDE Mg. group per
Product
(a’)
(b’) (c‘)
(d’)
Amino, N
Carboxyl. N
5.40 5.61 5.32 5.30
1.120 0.101 0.023 0.019
Mg. of element (or group) per 10 mg. preparationTotal, N C H I
10.8 10.2 10.8 10.7
28.4 27.0 27.6 27.6
5.5
gg: }
5.3 5.2
49.9 50.0
5.3
}
100 mg. preparate
Remarks
Soluble in water Transparent brittle film, soluble in water
after hydrolyiis Amino, Carboxyl, N N
..
...
10.1 10.6
5.0 5.5
10.7
5.4
tions with a small percentage of free amino-N samples of €-carbobenzoxy-a-carboxyl-1-lysine anand carboxyl-N. In accordance, Table I1 shows hydride containing different amounts of water thht products obtained on reduction of the prep- was measured a t 102’ (cf. Fig. 1). From these experiments, details of which are arations prepared by heating purified and thoroughly dried e-carbobenzoxy-a-carboxy-lysine an- given in the experimental part, it became obvious hydride, contain small amounts of free lysine. that water acts as a catalyst during the polymeriSome of the bulk products, (cf. b’c’d’, Table 11) zation of ecarbobenzoxy-a-carboxyl-1-lysine anobtained on reduction of the corresponding poly- hydride. Catalytic action, but to a smaller excarbobenzoxylysine preparations, were totally tent, was found with methanol. This conclusion hydrolyzed by boiling with 20% hydrochloric is in agreement with the findings of several preacid for twenty-seven hours. In the hydrolyzate, vious authors,s who found that N-carboxyl anpractically a quantitative yield of lysine was hydrides of various amino acids, while relatively stable in dry atmosphere, undergo rapid polyfound. The loss in weight of highly purified and thor- merization when exposed to moist air. oughly dried c-carbobenzoxy-a-carboxyl-1-lysine By taking into account the previously discussed anhydride during polymerization was measured. precautions, we could obtain poly-carbobenzoxyIt was found that this loss is equivalent to 98- lysine polymers and poly-lysines virtually free of 100~o of the theoretical amount of carbon dioxide monomer and possessing a considerable chain length. which could evolve on polymerization. In the following, we describe in some detail the The presence of amino nitrogen in the preparations described in Table I and the fact that the preparation and properties of one such polymer. e-Carbobenzoxy-a-carboxyl-lysine anhydride amino N content of these preparations is considerably higher than that of the corresponding recrystallized six times, and dried in high vacuum carboxyl-N’s seems to indicate that water plays a (lo-* mm.) over phosphorus pentoxide a t 60’ for certain role in the carbon dioxide cleavage of c- three hours, in an apparatus equipped with a earbobenzoxy-a-carboxyl-1-lysine anhydride, and liquid air trap, yielded on heating to 105’ in high vacuum (lo-’ mm.) for one hour, a transparent its polymerization. glassy hard polycarbobenzoxy-lysine. This polymer is insoluble in water and dissolves in hot glacial acetic acid. It contains free terminal amino groups determinable by Van Slyke’s manometric method. Assuming that the above polymer is a mixture of poly-carbobenzoxy-lysine peptides of various chain lengths, an average chain length of 32-lysine units was calculated from the values of free terminal amino-N. The carbobenzoxy groups of the poly-carbobenzoxylysine (aaverage = 32) were removed by reduction with phosphonium iodide according to the procedure used by Harington and Meads; attempted reductions by the usual catalytic methods were unsatisfactory. Poly-lysine hydriodide 10 30 50 70 90 110 (111) thus obtained, is very readily soluble in waTime in minutes. Fig. 1.-Per cent. of theoretical carbon dioxide, evolved ter, gives positive ninhydrin and biuret reactions, on polymerization of ccarbobenzoxy-acarboxyl-lysine and negative picric acid test. The latter negative anhydride versus time at 102’: 0 , monomer kept over test indicated the absence of lysine anhydride in water for forty minutes; 0,monomer dried over phos- the poly-lysine hydriodide polymer obtained. By making use of the ninhydrin-carbon dioxide phorus pentoxide. method for determination of free amino acids it In order to evaluate the role of water in our was found that (I11 n average = 32) contains a case, the rate of carbon dioxide evolution of two (8) Harington and Mead, Baol. Chem. J . , 99, 1603 (1935).
June, 1948
POLY-CONDENSATION OF (Y-AMINO ACIDDERIVATIVES : POLY-LYSINE
2097
very small amount of free lysine (0.2 mg. free ly- nitrophenyl) -lysine and e-2,Pdinitrophen yl-lysine sine per 100mg. (111)). were carried out colorimetrically according to Elementary analyses of poly-lysine hydriodide Sanger. Sanger found that during acid hydrolysis (n average = 22) arid ratio of amino nitrogen to of 2,Pdinitrophenyl derivatives of &-aminoacids, total nitrogen (found to be 1to 2) are in agreement partial decomposition occurs. This is taken into with the formilla suggested. On hydrolysis, poly- account in the evaluation of the amount of the terminal amino acids with free amino groups in lysine, yields lysine quantitatively. Independent support for the proposed consti- the insulin molecule. In order to determine the tution for poly-lysine hydriodide (naverage = 32) percentage decomposition of the two dinitrophenyl was obtained by using the procedure worked out lysine derivatives, under the hydrolytic conditions by who found that 2,4-dinitrofluoroben- applied in the present case, parallel experiments zene reacts with the free amino groups of a-amino were carried out with the corresponding 2,Pdiacids, peptides and proteins in the presence of so- nitrophenyl-lysine derivatives obtained synthetidium bicarbonate a t room temperature with cally. It was found that on boiling with 50% (w/ elimination of hydrogen fluoride. The dinitro- w) sulfuric acid for ten hours, 38% of a,c-di-(2,4phenyl-N-bond formed is relatively stable to acid dinitropheny1)-lysine and 25% of e-2,4-dinitrohydrolysis. Thus, in the case of insulin, he was phenyl-lysine were decomposed. After cortecting able, after coupling with 2,4-dinitrofluorobenzene for the decomposition of each of the 2,4-dinitroand following acid hydrolysis, to isolate and deter- phenyl-lysine derivatives during hydrolysis of 2,4mine quantitatively the 2,4-dinitrophenyl deriva- dinitrophenyl-poly-lysine,the amounts and ratio tives of those amino acids which in insulin bear between a,€-&-(2,4-dinitrophenyl)-lysine and e 2,4-dinitrophen 1-lysine were determined; 4.80 @e free amino groups. On coupling poly-lysine hydriodide (n average mg. of a,edi-&4-dinitrophenyl)-lysineJand 98 = 32) with 2,4-dinitrofluorobeene a t room mg. of e2,4-dinitrophenyl-lysineper 100 mg. of temperature, it yellow substance was obtained in 2,4-dinitrophenyl-poly-lysine,were found. The almost quantitative yields. The low value of ratio of the two compounds as determined in the amino N (Van Slyke 0.03%) in the polymer (IV) hydrolysate was therefore: 31 moles of e2,4-diindicates the blocking of all amino groups by 2,4- nitrophenyl-lysine per 1 mole of aJe-di-(2,4-dinidinitrophenyl groups. Dinitrophenyl-poly-lysine tropheny1)-lysine. The above data indicate that poly-lysine hydri(IV) was hydrolyzed in 50% (w/w) sulfuric acid during ten hours. The acid hydrolysate contains odide from which the 2,4dinitrophenyl-poly-lysine the a,e-di-(2,4-dinitrophenyl)-lysine and e-dinitro- was obtained, is built up on the average of 31 lyphenyl-lysine. The former is derived from the sine units. This conclusion is in satisfactory terminal lysine units of the poly-lysine containing agreement with the average chain length ascribed two (a and e) free amino groups, the latter from to the above poly-lysine hydriodide from the the other lysine units containing only one (e) free value of the amino-N of the poly-carbobenzoxylysine from which it had been derived by reducamino group. The a,e-di-(2,4-dinitrophenyl)-lysine contained tion. It must be emphasized, however, that the conin this hydrolyzate was quantitatively extracted with ether, while the c-2,4-dinitrophenyl-lysine siderable destruction of both dhitrophenyl-lysine remained in the aqueous acid solution. Both ly- derivatives during hydrolysis on the one hand, and sine derivatives were purified chromatographi- the low amino nitrogen content of poly-carbobencally; a,e-di-(2,4-dinitrophenyl)-lysineby using zoxy-lysine on the other, may lead to some uncerbutanol-chloroform 1%as developing solvent and tainty in the @ n a t i o n of the average chain e-2,4-dinitrophenyl-lysineby using methyl ethyl length. Nevertheless, the agreement between the ketone-ether (66%) as developing solvent. Wa- two pnalytical methods justifies, in our opinion, ter was the stationary phase in both cases. The R the drawing of a conclusion concerning the order values found for cr,e-di-(2,4-dinitrophenyl)-lysine of magnitude of the average chain length of the and e-2,4-dinitrophenyl-lysineobtained from the poly-lysine derivatives synthesized. From poly-lysine hydriodide (naverage = 32), acid hydrolyzate, were 0.6 and 0.2, respectively, under our cmditions. The same values were ob- the picrate, picrolonate, hydrochloride and beatained for the corresponding substances prepared zoyl derivatives were prepared. An indication for t h k presence of polymers of from 1-lysine. When each of the 2,4-dinitrophenyl-lysine derivatives, obtained from the various chain lengths was found by carrying out a polymer hydrolysate, was mixed with the corre- rough fractionation experiment. Poly-carbobensponding synthetic substance, and each of the zoxy-lysine, containing a very small amount of mixtures developed chromatographically by the monomeric lysine derivatives, yielded three fracsuitable developing solvent only one yellow band tions which differed in terminal amino nitrogen was obtained which in each case showed the char- content. acteristic R value. Preliminary enzymatic experiments were carThe quantitative estimations of a,c-di-(2,4-di- ried out with poly-lysine and lysine anhydride. It was found that poly-lysine is split by glycerol (9) Sanger, Amrhcrn J , $9, 507 (194.5)
2098
EPHRAWKATCHALSKI, ISAACGROSSFELD AND MAXFRANKEL
extract of pancreatin as well as by crystalline trypsin. About 50% of the peptide bonds were split. Lysine anhydride is not split by pepsin.
VOl. 70
in high vacuum (lo-' mm.) at 60" for three hours. The temperature was then elevated to 105'; carbon dioxide evolution started at once with the melting of the anhydride. After one hour no further gas evolution was observed; a transparent, glassy hard residue remained in Experimental the reaction vessel. The poly-carbobenzoxy-lysinepreparation thus obtained Preparation of e-Carbobenzoxya-carboxyl-1-lysineAnhydride.-1-Lysine was prepared from the red corpuscles is insoluble in water, ether or toluene. It is soluble in of ox blood according to Ricelo; from it e-carbobenzoxy- hot glacial acetic acid, and slightly soluble in hot alcohol. acarboxyl-lysine anhydride was prepared according to Part of the polymer precipitates from its solution in acetic Bergmann, Zervas and Ross.' The anhydride was re- acid on cooling, and from the supernatant solution a crystallized several times from ethyl acetate and petroleum further precipitate of polymer may be obtained on adding ether, and the purified starting material used at once for water, On heating the mixture of poly-carbobenzoxy-lysine polymerization experiments. The freshly purified prodoin water with ninhydrin for half an hour, the polymers uct dissolves readily in ethyl acetate, and its m. p. is 100 Estimation of Carbon Dioxide from e-Carobobenzoxy- turn deep blue, while the water remains almost colorless. The analytical data obtained correspond to a polymer carboxyl-lysine Anhydride on Heating to 102 .--1.140 g. of twice reaystalliied e-carbobenzoxy-a-carboxyl-lysine having formula (11) with an average chain length n = 32. Anal. Calcd. for (11) (n-average = 32 units): C, anhydride dried over phosphorus pentoxide in vacuo was kept at 102' for two hours. Carbon dioxide evolution 64.0; H, 6.9; N, 10.6; amino N, 0.17. Found: C, started on melting and a transparent, glassy p o l ~ e r 63.9: H. 7.1: N. 10.3: amino N, 0.17. The total nitroresidue was obtained. The weight of polymer residue gen .det&&ations, in this case as in other analytical was found to be 0.979 g. The weight of carbon dioxide data given in this paper, were carried out by the micro Dumas method. evolved (161 mg.) represents 98.5% of the theoretical. Rate of Carbon Dioxide Evolution at 102' from ePoly-lysine Hydriodide (n average = 32).-One gram Carbobenzoxya-carboxyl-lysine Anh dride (a) Kept of the above poly-carbobenzoxy-lysine (n average = 32) over Phosphorus Pentoxide; (b) dept over Water.was dissolved in 25 ml. of hot glacial acetic acid and the The rate of carbon dioxide evolution at 102' was measured solution kept at 50', while a stream of dry hydrogen was in an apparatus similar to that described by Hinshel- passed through it; 4 g. of phosphonium iodide was added anhydride in portions of about 1 g. during one to one and one-half wood." e-Carbobenzoxya-carboxyl-lysine twice recrystallized was dried over phosphorus pentoxide hours. The reduction of the carbobenzoxy groups was i n uacuo during two days at room temperature. One accompanied by a strong evolution of carbon dioxide. sample from the dried preparation was put at once into During the reduction, the phosphonium iodide dissolved, Hinshelwood's apparatus, the apparatus sealed, and and a voluminous precipitate formed on the walls of the placed in a constant temperature bath at 102'. The vessel. At the end of the reaction, the clear liquid was rate of carbon dioxide evolution was calculated from the decanted off and the residue washed several times with dry manometric readings of the apparatus. ether and dissolved in 2 ml. of water; to this solution The second sample of the dried c-carbobenzoxy-a- about 5 ml. of absolute alcohol and 70 ml. of ether were carboxy-lysine anhydride, weighing 67.85 mg., was put added. After standing overnight in the ice box, the superinto a desiccator over water for forty minutes. After natant fluid was decanted from the viscous material which this period i t was found to have gained in weight 0.870 had separated out. The latter was then dissolved in mg. The wet product was put into Hinshelwood's ap; several ml. of water, and the solution filtered and evaporparatus and the rate of carbon dioxide evolution at 102 ated to dryness in a vacuum desiccator over sulfuric acid measured as above. and sodium hydroxide. The transparent, solid film-like A comparison of the rate of carbon dioxide evolution polymer thus obtained was washed several times with in both cases is given in Fig. 1. ether and dried. It was obtained in almost quantitative The increase in rate of carbon dioxide evolution in the yield. Poly-lysine hydriodide was dried in a micr! presence of water may be at least partly explained by the vacuum desiccator over phosphorus pentoxide at 80 catalytic action of water on the polymerization of c- before analysis. carbobenzoxy-a-carboxyl-lysine anhydride according to Anal. Calcd. for (111) (n average = 32): C, 27.7; the first step of the general scheme. Nevertheless, it H, 5.0; N, 10.7; amino N, 5.5; I, 50.2. Found: C, should be borne in mind that water may open the an- 27.6; H, 5.2; N, 10.7; amino N, 5.3; I, 50.0. The hydride bond of the N-carboxyl anhydride, and thus lead amino N value was obtained after shaking the polymer to carbobenzoxy-lysine with carbon dioxide evolution. solution with HNOI for five minutes; this value was not Regarding the latter possibility, it should be remarked, altered on further shaking for half an hour. that for quantitative transformation of c-carbobenzoxyPoly-lysine hydriodide dissolves very readily in water a-carboxyl-lysine anhydride into e-carbobenzoxy-lysine, water in an amount of 5.9% of the weight of the anhydride a n d i t s aqueous solution gives a positive ninhydrin reis needed, whereas only 1.3% of water was taken up by action and a strong biuret reaction. A negative Abdere-carbobenzoxy-a-carboxyl-lysineanhydride in our case. halden test was obtained on heating with an alkaline The fact that this amount of water is sufticient to shorten solution of picric acid. Poly-lysine hydriodide does not dissolve in the usual the time of cleavage, involving the loss of 95% of the theoretical amount of carbon dioxide in c-carbobenzoxy- organic solvents. Search for Free Lysine in Poly-lysine Hydriodide (n a-carboxyl-lysine anhydride, from ninety-five minutes to twenty minutes,, justifies the conclusion that water acts average = 32).-The amount of free lysine in the polyunder our expenmental conditions mainly as polymeriza- lysine hydriodide described above was determined by the tion catalyst. This is borne out also by the polymeric ninhydrin-carbon dioxide method a t PH 2.5. In 100 mg. of polymer product described in the previous section 0.2 properties of the reaction products. Preparation of Poly-carbobenzoxy-lysine (n average = mg. of free lysine was found. Total Hydrolysis of Poly-lysine Hydriodide (n average 32 Carbobenzoxy-lysine Units).-The starting c-carbo= 32).--15.9 mg. of dried poly-lysine hydriodide was benzoxy-a-carboxyl-lysineanhydride for the polymerization, was recrystallized six times from ethyl acetate and dissolved in 4 ml. of 20% hydrochloric acid and the petroleum ethcx. The purified product was introduced solution boiled under reflux for twenty-four hours. The immediately into a glass vessel connected to an apparatus acid hydrolyzate was neutralized with sodium hydroxide with phosphorus pentoxide and a liquid air trap and dried and brought to 15 ml. In 2 ml. of the final solution the amount of carboxyl N was determined by using the ninhydrin-carbon dioxide (10) Rice, J B i d C h e m , 131, 1 (1941). method'; in the other 2 ml., the total free amino N (Van (11) Hinehe1wood.J Ckern Soc ,117, l5G (1920).
.
June, 1948
POLY-CONDENSATION OF
a-AMINOACIDDERIVATIVES : POLY-LYSINE
Slyke’s manometric method-on shaking half an hour with nitrous acid-was determined. From the data obtained, the total amounts of carboxyl N and amino N in hydrolyzate were calculated. The amount of these groups per 100 mg. starting material are given below. Anal. Calcd. for hydrolysis of 100 mg. poly-lysine hydriodide ( n average 32). Carboxyl-N 5.50 mg.; amino N, 10.00 mg. Found: carboxyl N, 5.4 mg.; amino N, 10.7 mg.; on hydrolysis of 100 mg. of starting material. The analytical data show that the total amount of amino-N is equal to twice that of carboxyl N and thus indicates the presence of free lysine in the hydrolyzate. Furthermore, it is proved that the starting poly-lysine hydriodide is built up quantitatively of lysine units. Preparation of 2,4-Dinitrophenyl-poly-lysine.-l50 mg. of poly-lysine hydriodide ( n average 32) and 3.1 g. of sodium bicarbonate were dissolved in 30 ml. water; 30 ml. of ethanol and 5 ml. of 2,4-dinitrofluorobenzenewere then added and the mixture mechanically shaken for two hours. A large excess of 2,4-dinitrofluorobenzene was necessary in order to assure complete coupling of the large number of free amino groups in poly-lysine hydriodide with the 2,4-dinitrophenyl reagent. The yellow precipitate of 2,4-dinitrophenyl-poly-lysinewas centrifuged and washed several times with water and ethanol and dried in a vacuum desiccator. 2,4-Dinitrophenyl-poly-lysine is a yellow powder, insoluble in water, alcohol, ether, and glacial acetic acid. It is soluble in concentrated sulfuric acid, and can be precipitated from it by addition of water. Anal. Calcd. for 2,4-dinitrophenyl-poly-lysine (n average = 32): amino N, 0.0; N, 18.9. Found: amino N,0.03; N, 19.0. The amino N value found indicates that practically all of the amino groups of poly-lysine reacted with 2,4dinitrofluorobenzene. Hpdrolysis of 2,4-Dinitrophenyl-poly-lysine; Identificahon and Estimation of a,e-Di-(2,4-dinitrophenyl) - and c-2,4-Dinitrophenyl-lyaine Fomed.-2,4-Dinitrophenylpoly-lysine is not hydrolyzed quantitatively in 2070 hydrochloric acid even on boiling for extended periods, as clumps of polymer settle to the bottom of the vessel and do not enter into solution. We thus looked for another acid in which the polymer would dissolve more readily before hydrolysis. It was found that total hydrolysis of 2,4dinitrophenyl-poly-lysinecan be carried out in 50yo (w/w) sulfuric acid; the following hydrolytic experiment was therefore carried out in this medium. Twenty mg. of 2,4dinitrophenyl-poly-lysine was boiled under reflux for ten hours in 20 ml. 50% (w/w) sulfuric acid. After cooling, the solution was extracted five times with ether. The first two ether extracts showed a clear yellow color which could be attributed to the presence of a,c-di-(2,4-dinitrophenyl)-lysine in the hydrolyzate. The ether extracts were washed with a small amount of water and the washings returned to the original aqueous solution. The collected ether extracts were reduced to dryness, and passed through an ether column prepared from 2 g. of silica, and again evaporated to dryness, and developed on a 1% butanol-chloroform column. There was a red band that moved rapidly (R = 1.5) and a yellow band which moved much more slowly (R = 0.6). After repeating the separation between the two bands on a 3 g. of 1% butanol-chloroform column, the red band was decanted and not explored further, as it seemed to represent a breakdown product of hydrolysis. The R of the yellow band was that found for synthetic a,e-di-(2,4-dinitrophenyl) -lysine prepared from lysine and 2,4-dinitrofluorobenzene. Furthermore, in a parallel hydrolysis experiment, it was found that on mixing the fraction of yellow band with synthetic a,e-di-(2,4-dinitrophenyl)lysine, evaporating to dryness and developing with 1% butanol-chloroform one band only with the anticipated R was formed. The solution of the yellow band is thus identified as thc solution of a,c-di-(2,4-dinitrophenyl)-lysine. 5
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2099
I n order to estimate the a,r-di-(2,4-dinitrophenyl)lysine formed by the hydrolysis of 2,l-dinitrophenylpoly-lysine quantitatively, the fraction of yellow band mentioned above was run out, taken to dryness, the residue dissolved in 50 d.of chloroform and estimated colorimetrically, with a photoelectric absorptiometer after plotting the standard curve of synthetic a,e-di-(2,4dinitropheny1)-lysine in chloroform; 0.596 mg. was found. The acid solution, after extraction with ether, was diluted to 500 ml. with water, and the sulfuric acid removed quantitatively by means of barium chloride. The precipitate of barium sulfate was filtered off and washed several times with water. The combined filtrate and washings were made up to lo00 ml. A 100-ml. sample from this solution was withdrawn and evaporated to dryness. The residue was passed through a 66% methyl ethyl ketone ether column on silica gel. One yellow band 0.2. The same R was obtained was formed having R when synthetic ~-2,4dinitrophenyl-lysineprepared according to Sanger was passed through this column, and when the residue from aqueous solution of hydrolyzate, described above, in another hydrolytic experiment, was mixed with synthetic c-2,4-dinitrophenyl-lysine and the mixture passed through a 66% methyl ethyl ketone ether column. The presence of c-2,4-dinitrophenyl-lysine in the aqueous solution of hydrolyzate is thus proved. In order to determine quantitatively the amount of e-2,4-dinitrophenyl-lysine obtained, the yellow band in the original column was run out, taken to dryness and made up to 50 ml. with N hydrochloric acid. The amount of e-2,4-dinitrophenyl-lysinein this solution was estimated colorimetrically. A total amount of 14.7 mg. of c-2,4dinitrophenyl-lysine was found in the lo00 ml. of aqueous solution. In order to estimate the amount of a,c-di-(2,4-dinitrophenyl) -lysine and e-2,4-dinitrophenyl-lysine,decomposing under the conditions prevailing in the hydrolysis of 2,4-dinitrophenyl-poly-lysine,weighed amounts of synand r-2,4-dinitrothetic a,e-di-(2,4-dinitrophenyl)-lysine phenyl-lysine were kept in boiling 50% (w/w) sulfuric acid for ten hours. In the first case, the amount of a,r-di(2,4-dinitrophenyl) -lysine and in the second case, the amount of c-2,4-dinitrophenyl-lysine which remained unaffected by the acid were determined as above. A breakdown of 25% of c-2,4-dinitrophenyl-lysineand a breakdown of 38% of a,c-di-(2,4dinitrophenyl)-lysine were obsesved under the hydrolytic conditions used. If the values obtained on the hydrolysis of 2,4-dinitrophenyl-poly-lysine are corrected by the last data for the breakdown of c-2,4-dinitrophenyl-lysineand a,r-di-(2,4dinitropheny1)-lysine, the following yield from 100 mg. of 2,4-dinitrophenyl-poly-lysine of a,adi-(2,4-dinitrophenyl) -lysine and c-2,4-dinitrophenyl-lysine, are obtained: 4.80 mg. of a,edi-(2,4-dinitrophenyl)-lysine and 98.0 mg. of c-2,4-dinitrophenyl-lysine. Although as already remarked by Sanger, “the correction may not be strictly valid, as the stability of the derivatives while still condensed in the protein or in peptide split products may not be the same as that found for the derivatives themselves,” it seems that at least a good approximation for the real estimation of the two 2,4dinitrophenyl-lysine derivatives is thus obtained. It can be seen that 31 moles of e-2,4dinitrophenyllysine per 1 mole of a,tdi-(2,4dinitrophenyl)-lysine was obtained in the hydrolyzate. As pointed out previously, these data are in fair agreement with the suggested structure for poly-lysine hydriodide (n average 32). Proof for the Practical Absence of a,c-Di-(2,4-dinitrophenyl) -lysine, in 2,4-Dinitrophenyl-poly-lsine.-In order -lysine is to prove that the a,e-di-(2,4dinitropgenyl) derived entirely from the original terminal lysine units of the 2,4-dinitrophenyl polymer, and does not partly originate from the coupling of 2,4-dinitrofluorobenzene with any free lysine monomer in the poly-lysine hydriodide (n average = 32), the following experbent was carried out. A sample of 30 mg. of 2,4dinitrophenyl-poly-lysine
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2100
EPHRAIM KATCHALSKI, ISAACGROSSFELD AND MAXFRANKEL
was dissolved without heating in 10 ml. of concentrated sulfuric acid, 20 ml. of water was slowly added under strong cooling, and the mixture shaken vigorously with ether. No yellow color indicating the presence of a,cdi-(2,4-dinitrophenyl)-lysine appeared in the ether extract. On the other hand, when a sample of 2,4dinitrophenylpoly-lysine dissolved in 50% (w/w) sulfuric acid was boiled for about thirty minutes and the mixture then extracted with ether, the formation of small amounts of a,a-di-(2,4-dinitrophenyl)-lysine due to hydrolysis was ent from the faint yellow color of the ether extract. ap&-lysine Picrats.-lOO mg. of poly-lysine hydriodide (naverage = 32) was dissolved in 2 ml. of hot water, and 2 ml. of hot saturated solution of picric acid added. After standing overnight in the ice box, the yellow picrate was filtered off and washed several times with cold water and ether. Yield was 85% of the theoretical. The groduct was thoroughly dried in vacuum desiccator at 100 before analysis. Anal. Calcd. for poly-lysine picrate (n average = 32): C, 40.5; H, 4.2; N, 19.5. Found: C, 40.1; H, 4.4; N, 19.4. The picrate is soluble in hot water, slightly soluble in alcohol but insoluble in ether and benzene. Poly-lysine Picrolonate.-l00 mg. of poly-lysine hydriodide (n average = 32) was dissolved in 2 ml. of water, and 5 ml. of hot saturated solution of picrolonic acid in water added. A voluminous precipitate separated a t once. After standing overnight in the icebox, the precipitate was filtered and washed several times with water and ether. The material was thoroughly dried in a vacuum desiccator a t 100" before analysis. Anal. Calcd. for polylysine picrolonate (n average = 32): C, 49.0; H, 4.9; N, 21.4. Found: C, 48.6; H, 5.1; N, 21.1. I t is sparingly soluble in water and insoluble in the usual organic solvents. Yenmyl-poly-lysine.-l00 mg. of poly-lysine hydriodide ( n average = 32) was dissolved in 2 ml. of water, 400 mg. of sodium bicarbonate added and the mixture cooled to 0". On further cooling, 0.5 ml. of benzoyl chloride in five portions was added with vigorous shaking. After forty-five miniites, the mixture was acidified to congo red with concentrated hydrochloric acid, and the white precipitate decanted and thoroughly washed with water, alcohol and ether. Benzoyl-poly-lysine was obtained in Soyqyield as a white powder. It is soluble in hot glacial acetic acid and concentrated sulfuric acid, but insoluble in ether, alcohol and water. For anaoysis the polymer was dried in vacuum desiccator at 100 . Anal. Calcd. for benzoyl poly-lysine (n average = 32): N, 11.86. Found: N, 11.60. Poly-lysine Hydrochloride.4.65 ml. of N hydrochloric acid was added to 150 mg. of poly-lysine picrate suspended in 5 ml. of water. . The mixture was shaken several times with ether to remove the picric acid liberated. An equal volume of alcohol was then added to the aqueous solution and the mixture dried at room temperature in a vacuum desiccator over sulfuric acid and sodium hydroxide. The remaining hydrochloride was washed several times with dry ether and dried in vacuo. The yield was quantitative. The hydrochloride was further dried at 80" in vacuo over phosphorus pentoxide before analysis. Anal. Calcd. for poly-lysine hydrochloride (n average = 32) : C1,22.3. Found: C1,22.0. Poly-lysine hydrochloride is very soluble in water and gives a strong violet biuret reaction. The ninhydrin reaction is positive, and when heated with an alkaline solution of picric acid, no red coloration is obtained. Polylysine hydrochloride is insoluble in usual organic solvents. Preliminary Experiment in the Fractionation of a Polycarbobenzoxy-lysine Preparation.-This experiment was carried out with the material described under Table IC;
Vol. 70
this preparation contains a very small percentage of carboxyl-N indicating a very small percentage of "monomers." 100 rng. of polymer was dissolved in 5 ml. of hot glacial acetic acid. The solution was allowed to cool and the precipitate (fraction 1) filtered. To the filtrate an equal amount of water was added and the turbid mixture formed allowed to stand overnight in the icebox. The white precipitate (fraction 2) was filtered off and the filtrate concentrated in w u o to dryness (fraction 3). I n the three fractions the free amino N (Van Slyke) was determined. Fraction
1 2
3
Weight of fraction, mg.
18 63 19
Amino N, %
0.18
.23 .30
Discussion The structure suggested for the polymeric derivatives obtained from ecarbobenzoxy-a-carboxyl-lysine anhydride (cf. scheme) calls for proof of the presence of peptide bonds and free a-amino and a-carboxyl terminal groups. The presence of the assumed 420"bonds is indicated by the positive biuret reaction, and proved by the formation of the expected equivalent amounts of a-amino and u-carboxyl groups on hydrolysis of poly-lysine hydriodide. The peptide structure of poly-lysine is also supported by the fact that crystalline trypsin causes cleavage of this polymer. This enzymatic result is in agreement with the findings of Bergmann and Fruton,'* that a typical trypsin substrate has to contain lysine or arginine residues. Furthermore, the method of synthesis of polylysine and the fact that it yields on hydrolysis lysine quantitatively, leave little doubt that the polymer is built up of lysine residues bound by normal peptide bonds. The positive ninhydrin reaction in poly-lysine hydriodide and poly-carbobenzoxy-lysine indicates the presence of a-amino groups. The presence of the a-amino end-group in poly-carbobenzoxy-lysine is proved by the Van Slyke manometric amino nitrogen determination, while the presence of the same terminal group in poly-lysine hydriodide is proved by the isolation of u,a-di-(2,4 dinitrophenyl)-lysine from 2,4-dinitrophenyl-plylysine. The formation of the terminal groups can be explained by the observed catalytic action of water on the polymerization of a-carbobenzoxya-carboxyl-lysine anhydride. The above facts support our view that the polylysine derivatives described in this paper represent mixtures of straight chains of poly-lysine peptides probably of various chain length; nevertheless the presence of high rings in the polymeric mixtures cannot be excluded. In connection with our previous work on the polycondensation of esters of a-amino acids,l+? the absence of diketopiperazine in the product obtained on polymerization of e-carbobenzoxy-ucarboxyl-lysine anhydride is of interest. While (12) Bcrgmann and Fruton, "Advances in Enzymology," I. 73 (1941)
June, 1948
PHOSPHORYLATION OF PROTEINS
2101
the methyl ester of e-carbobenzoxy-lysine yields tions, a poly-carbobenzoxy-lysine was obtained on heating mainly e,c'-dicarbobenzoxy-lysine an- with an average chain length of 32-carbobenzoxyhydride,8 the corresponding N-carboxyl anhydride lysine units. yields a polymer without any diketopiperazine. Poly-carbobenzoxy-lysine (11) (naverage = 32) This difference may be connected with the fact yields on reduction with phosphonium iodide polythat polycondensation of a-amino acid esters pro- lysine hydriodide (111) .( average = 32). ceeds slowly, while the polymerization of the corPoly-lysine hydriodide (n average = 32) conresponding N-carboxyl anhydrides is rapid. Thus tains practically no free lysine or lysine anhydride. the methyl ester of e-carbobenzoxy-lysine con- On hydrolysis it yields lysine quantitatively. denses to €,e'-di-carbobenzoxy-lysineanhydride a t The following derivatives were prepared from 105' over a period of several days, while the poly- (111) (n average = 32): picrate, picrolonate, hymerization of e-carbobenzoxy-a-carboxyl-lysine drochloride, benzoyl and 2,4-dinitrophenyl-polyanhydride is accomplished under the same condi- lysine. tions within an hour. The suggested formula for poly-lysine (n averSummary age = 32) is supported by the analytical data, and cCarbobenzoxy-a-carboxyl-lysine anhydride by the fact that 2,4-dinitrophenyl-poly-lysine(n (I) when heated to 102-105' evolves carbon diox- average = 32) yields the expected amounts of ide and yields a polymeric preparation to which a,e-di-(2,4-dinitrophenyl)-lysineand e-2,4-dinitrothe structure of poly-carbobenzoxy-lysine (11) is phenyl-lysine on hydrolysis. Independent support for the presence of pepascribed. The products of polymerization of (I) obtained tide bonds in (111) is given by its cleavage with crystalline trypsin. under various conditions were studied. On polymerization of (I) under special precauRECEIVED DECEMBER 9, 1947
[CONTRIBUTION FROM THE WESTERN REGIONAL RESEARCH LABORATORY']
Phosphorylation of Proteins with Phosphoric Acid Containing Excess Phosphorus Pentoxide B Y ROBERTE. ~ R R E L HAROLD , S. OLCOTT AND HEINZFRAEN-KEL-CONRAT When proteins are treated with cold concentrated sulfuric acid, the principal reaction is a transformation of the aliphatic hydroxyl groups to half-esters of sulfuric acid.*J The present study was undertaken in order to determine whether analogous reactions occur when proteins are treated with phosphoric acid containing excess phosphorus pentoxide. Levene and Schormiiller* had used such a reaction mixture for the preparation of o-phosphoric acid esters of serine, hydroxyproline and serine anhydride in small yield, and Plimmer,busing the same medium a t elevated temperatures, had duplicated their findings and also reported the preparation of o-phosphoric acid esters of tyrosine, threonine and isoserine. In general, the reaction was carried out by permitting a mixture of the material to be treated and the phosphoric acid-phosphorus pentoxide reagent to stand for three days a t room temperature in a dry atmosphere (desiccator). The product
was isolated by pouring the reaction mixture over cracked ice, neutralizing, and dialyzing, first against ion-free water, then against 10% sodium chloride, and finally against distilled water until free of inorganic phosphates. Recoveries ranged from 70 to 100% based upon nitrogen analyses. The extent of reaction was estimated from the phosphorus-to-nitrogen ratio of the product. Of the many polar groups in proteins available for reaction, only the aliphatic hydroxyl groups of serine, threonine and hydroxyproline, and to some extent the aromatic hydroxyl group of tyrosine were found to bind phosphorus in a stable mannera6 The basic groups and the peptide bonds, however, appear to be responsible for an additional amount of phosphate, retained during dialysis against water but liberated by high salt concentration. Part of the peptide bonds were labile to
(1) Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. Article not copyrighted. (2) Reitz, Ferrel, Fraenkel-Conrat and Olcott, THIS JOURNAL, 68, 1024 (1946). (3) The product obtained from wheat gluten was gel-forming and appeared to have possible industrial significance (Reitz, Ferrel and Olcott, Ind. En#. Chcm., 86, 1149 (1944)). (4) Levene and Schormiiller, J . Bid. Chcm., 106, 547 (1934); 106, 695 (1934). (5) Plimmer, Biochcm. J . , 116, 461 (1941).
(6) This specificity contrasts with the non-specific action of other phosphorylating agents which are known to react with amines, guanidyl compounds, etc.. as well as with alcohols and phenols. Mayer and Heidelberger' phosphorylated horse-serum albumin in alkaline solution with phosphorus oxychloride. The derivatives contained 2 4 % phosphorus, approximately half of which was accounted for by reaction with the amino groups. The reaction of egg albumin under similar conditions was described by Heidelbuger, cf a1.a
(7) Mayer and Heidelberger, TEISJOURNAL, 68, 18 (1946). ( 8 ) Heidelberger, Davis and Treffus, ibid.. 611, 498 (1941).
